Chimeric cell-targeting pathogenic organism and method of therapeutic use

ABSTRACT

The invention chimeric organism comprises a chimeric surface integrin-like fusion protein in which the I domain has been replaced by an antibody fragment that binds a disease-associated antigen on a cell. Binding of the antibody fragment to the disease-associated antigen triggers virulent transformation of the chimeric pathogenic organism so as to cause the organism to infiltrate the target cell with specificity. Preferably, the chimeric organism is a chimeric pathogenic  C. albicans  having an INT1 fusion protein in which the I domain is replaced by an antibody fragment, preferably a single chain antibody, and in which expression of an iron transporter gene necessary for infiltration of a target cell is triggered under the control of a EFG1p response element. Binding of the antibody to the disease-associated antigen causes filamentous transformation in the chimeric pathogenic  C. albicans  and specific infiltration of target cells. The invention chimeric pathogenic organisms are used in therapeutic methods to specifically infiltrate and destroy diseased cells to which the antibody fragment binds while remaining non-pathogenic to normal cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/002,389 filed Nov. 30, 2001, now U.S. Pat. No. 6,638,756; whichclaims priority under 35 USC §119(e) to U.S. Application Ser. Nos.60/297,995 filed Jun. 13, 2001, now abandoned and 60/251,253 filed Dec.5, 2000, now abandoned. The disclosure of each of the prior applicationsis considered part of and is incorporated by reference in the disclosureof the application.

FIELD OF THE INVENTION

This invention relates to treatment of diseases characterized byproduction of cell surface markers using antibody-targeted compositions.More particularly, this invention relates to chimeric organisms thatexpress an antibody fragment and to the use of such chimeric organismsin treatment of diseases characterized by production of cell surfacemarkers.

BACKGROUND OF THE INVENTION

Many recent gene therapy approaches have exploited the specificity ofantibody binding to target cancer cell lines in order to deliver eitherdrugs or immune responses to an actual tumor location. Most cancer celllines misregulate cell surface proteins and polysaccharides, and arethus easily distinguished from normal somal cells by antibodies (R. E.Hawkins et al., Gene Therapy (1998), 5:1581-1583). It is apparent thatestablished carcinomas have successfully avoided activating the immuneresponse within their hosts. Direct attempts to rectify this byrecruiting the body's humoral immune response to tumors by injection ofmurine derived antibodies can unfortunately cause serious and even lifethreatening human anti-mouse responses (R. K. Jain et al., J Natl.Cancer Inst. (1989) 81:570-576 and D. Colcher et al., J. Natl. CancerInst. (1990) 82:1191-1197). In addition, the overall penetration ofantibodies into tumors is limited due to the high molecular weights ofthese molecules (K. A. Chester et al., Adv. Drug Delivery Rev. (1996)22:303-313).

In an attempt to limit both the size of the antibody and themouse-character of the antibody, single chain antibodies (scFvs) thatencapsulate the binding features of the Fv region of the antibodywithout the bulk of the native antibody sequence in the c1, c2, and c3domains have been developed. One methodology to generate scFvs involvestethering the antigen binding domains of V_(H) and V_(L) together usinga short flexible peptide linker (R. E. Bird et al., Science (1988)242:423-426). Another approach involves the generation de novo ofmolecular diversity, instead of generating monoclonal antibodies inmice. By using combinatorial antibody libraries on the surface offilamentous bacteriophage screened against immobilized antigen, a singlepolypeptide chain that is amenable to fusion with other proteins can begenerated (J. S. Huston et al., Proc. Natl. Acad. Sci. USA (1988)85:5879-5883; J. McCafferty, Nature (1990) 348:552-554; R. H. J. Begentet al., Nature Med. (1996) 2:979-984, reviewed in K. A. Chester et al.,Adv. Drug Delivery Rev. (1996) 22, 303-313). The scFvs obtained byeither methodology above show better tumor penetration, but therapeuticapplication is still in early stages (G. Reitmuller et al., Lancet(1994) 343:1177-1183). However, fusions between imaging agents and scFvshave found wide acceptance and extensive application in tumor imagingand radiochemotherapeutic delivery (see J. Bhatia et al., Cancer (1999)85:571-577 and A. M. Wu et al., Tumor Targeting (1999) 4:47-58 andreferences therein).

Antibody recognition has also been used to target cancer cells byincorporation of an scFv into the envelope protein of a retrovirus (S.J. Russell et al., Nuc. Acids Res. (1993) 21:1081-1085 and F. Martin etal., Human Gene Therapy (1998) 9:737-746). This targeting is modest, butoffers some promise, as has been demonstrated for certain types ofmelanoma (Martin 1998). In addition, adenovirus infection has been usedto allow the transient expression of tumor-targeting scFv fusionproteins in whole organisms with moderate success (H. A. Whittington etal., Gene Therapy (1998) 5:770-777). Unfortunately, low survivability ofadenoviruses carrying antibody generating expression vectors limitstheir impact.

The most promising therapeutic techniques relying on the specificity ofantibody binding focus on engineering T-cells that express antibodyfragments fused to surface proteins, and are thus directed to tumorsurfaces (recent work reviewed in F. Paillard, Human Gene Therapy (1999)10:151-153). Some of these T-cells are at present in clinical trials.Strategies used to date, however, have drawbacks, including limitedefficacy against established tumors, though demonstrating some slowingof tumor metastasis (R. P. McGuinness et al, Human Gene Therapy (1999)10:165-173). Limited effectiveness against established tumors may be dueto the inability of the T-cells to penetrate solid cell masses (Paillard1999). True protection against establishment of invasive carcinoma wasobtained only by coinjection of modified T-cells with the tumorogenicline. In clinical applications, this may permit stabilization andlocalization of established tumors, but not reductive treatment. Anotherpotential problem is that suicide signals T-cells use to induceapoptosis, like tumor necrosis factor I, are often not functionalagainst carcinomas. Even when they are effective, successful cancer celllines will rapidly adapt to apoptotic signals, and have even been knownto induce apoptosis in attacking T-cells (K. Shiraki, Proc. Natl. Acad.Sci. USA (1996) 94:6420-6425). In addition, T-cells bearing thesechimeras are assembled separately for each patient ex vivo due topossible MHC incompatibilities that could result in serious allergicreactions were T-cells from other humans introduced therapeutically.

Candida albicans is the most commonly isolated invasive fungal pathogenin humans. This organism is representative of several that switchbetween two major classes of morphology. The first morphology is theellipsoid blastospore. Like most yeast, C. albicans assumes thisarchitecture when growing non-pathogenically. Upon binding of C.albicans to mammalian tissues (i.e. via the I domain of the INT-1protein), the cell morphology switches to various filamentous forms,including germ tubes and hyphae, that are capable of aggressivelyinvading host tissue (reviewed by R. A. Calderone, Microbol. Rev. (1991)55, 1-20). Systemic infection of a vulnerable host by C. albicansresults in high levels of mortality. For example, more than 30% ofimmunocompromised HIV patients are systemically infected despiteappropriate treatment regimes. In addition, C. albicans infectioncommonly leads to death in premature infants, diabetics, and surgicalpatients. To date, the ability of this pathogenic organism to infectcells when the cell morphology switches to a filamentous form has notbeen utilized for therapeutic purposes, such as in cancer therapy.

Thus, the need exists in the art for new and better compositions andmethods of their use for treating various types of cancers and otherdiseases associated with production of an abnormal protein.

SUMMARY OF THE INVENTION

The present invention overcomes these and other problems in the art byproviding chimeric organisms having a chimeric surface integrin-likeprotein in which the I domain has been replaced by an antibody fragmentthat binds a disease-associated antigen on a cell. Binding of theantibody fragment to the disease-associated antigen on the cell triggersvirulent transformation of the chimeric pathogenic organism and allowsthe organism to infect the cell.

In one embodiment according to the present invention, there are providedchimeric pathogenic C. albicans modified to contain an integrin1 (INT1)fusion protein in which the I domain is replaced by an antibody fragmentthat binds to a disease-associated antigen on a diseased cell. Thechimeric C.albicans further contains a disabled wild-type highaffininity iron transporter (CAFTR) gene, and a DNA construct comprisinga wild-type CAFTR gene under the control of an enhanced filamentousgrowth protein (EFG1p) response element, wherein binding of the antibodyto the disease-associated antigen triggers expression of the CAFTR genein the DNA construct and filamentous transformation in the chimericpathogenic C. albicans.

In another embodiment according to the present invention, there areprovided methods for treating a disease associated with the presence ofcells having a disease-associated surface antigen in a subject in needthereof by administering to the subject a therapeutically effectiveamount of an invention chimeric pathogenic organism so as to causebinding of the antibody fragment to the disease-associated antigen onthe cells, thereby treating the disease by triggering infiltration ofthe chimeric pathogenic C. Albicans into the cells without substantialdamage to healthy cells.

In yet another embodiment, the present invention provides methods forgenerating a chimeric therapeutic organism from a pathogenic organismthat possesses in the wild-type an integrin-like protein with an Idomain. In the invention methods, the I domain in the integrin-likeprotein of the pathogenic organism is replaced with an antibody fragmentthat binds to a disease-associated antigen on a diseased cell. In thechimeric therapeutic organism, virulent transformation occurs uponbinding of the antibody fragment to the disease-associated antigen onthe cell.

A BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1—1 to 1-23 show the nucleotide sequence of the gene that encodesthe integrin-like INT1 protein C. Albicans (GenBank Accession #U35070)(SEQ ID NO:1).

FIG. 2 shows the nucleotide sequences of seven primers used inconstruction of the chimeric C. albicans of Example 1 (SEQ ID NOS:2through 8, respectively).

FIG. 3 is a schematic drawing showing human integrin structure (adaptedfrom M. J. Humphries, Biochem. Soc. Trans. (2000) 28:311-340).

FIG. 4 is a schematic drawing showing two pathways by which hyphaldevelopment in yeast is regulated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides chimeric pathogenic organisms derivedfrom wild type organisms wherein virulent transformation of the organismis controlled in the wild-type organism by binding of the I domain of asurface integrin-like protein to a cell. The invention chimeric organismcomprises a chimeric surface integrin-like protein in which the I domainis replaced by an antibody fragment that binds a disease-associatedantigen on a cell. Binding of the antibody fragment to thedisease-associated antigen triggers virulent transformation of thechimeric pathogenic organism so as to cause the organism to infiltratethe cell. Virulent invasion of the cell by the chimeric pathogeninhibits growth of the diseased cell.

The invention pathogenic chimeric organism represents a new approach toemploying otherwise pathogenic organisms to assist in disease treatment.Although the present invention is described for illustrative purposeswith reference to a reingeneered C albicans, suitable pathogenicorganisms in addition to C. albicans that can be engineered according tothe methods disclosed herein are pathogenic organisms that becomevirulent (e.g., switch to a filamentous invasive form) upon binding ofits integrin-like surface protein (i.e., a cell-cell communicationprotein) to a target on another cell and in which the binding domain ofthe surface protein can be replaced with a antibody fragment that bindsto a desired target cell associated with a disease state. Preferably thechimeric pathogen also is relatively harmless to mammalian cells untilbinding of the antibody fragment contained in its surface protein.

In higher eukaryotes, integrins are one of the most important classes ofsurface proteins responsible for intercellular communication (reviewedin F. G. Giancotti Science (1999) 285:1028-1032 and M. J. Humphries,Biochem. Soc. Trans. (2000) 28:311-340). Generally, integrins areheterodimers, each subunit of which consists of a cytosolic domain withone tyrosine used as a kinase regulatory site, a transmembrane domain,and four EFG-like repeats. As used herein, the term “integrin-likeprotein” refers to a cell-cell communication transmembrane protein thatcontains one or more of the above features.

There are various other domains on the integrin proteins, includingmetal binding MIDAS loops and β propeller domains. Notably, in nine ofthe fifteen human integrin I subunits, there is a protruding regionknown alternately as the IA, or the I domain, which appears to regulateintegrin targeting. This suggests that the absence or presence of the Idomain has little, if any, effect on the integrin's ability to transducesignals, but instead regulates which signals are transduced. The Idomain is the only region whose structure has been solvedcrystallographically (both bound to its target proteins and unbound).Based on these studies, it is believed that the I domain alone is indeedsufficient for binding to collagen (J. Emsley Cell (2000) 101:47-56).

In the invention chimeric organism, the endogenous binding region of thesurface integrin-like protein, which nonspecifically targets cells(e.g., those containing fibrinogen), is replaced with an antibodyfragment, such as a single chain antibody. As a result, rather thannonspecifically binding to any cell containing a binding site for theendogenous binding region, the invention chimeric pathogen binds withspecificity to cells that express the target antigen. Binding of thechimeric pathogen to a cell containing an epitope for the antibodyfragment triggers virulent invasion of the disease-associated cell.Other cells (e.g., healthy cells) are not bound by the chimericorganism. As a result, pathogenic infiltration of non-targeted cellsdoes not take place.

In one embodiment, the invention provides a chimeric pathogenic C.albicans comprising an INT1 fusion protein in which the I domain isreplaced by an antibody fragment that binds to a disease-associatedantigen on a cell. Preferably, the INT1 protein in the inventionpathogenic organism is a fusion protein in which a single chain antibodyreplaces the native I domain. The nucleotide sequence encoding INT1 isshown in FIGS. 1A-W (SEQ ID NO:1 herein). Construction of such achimeric INT1 is described in Example 1 below.

As used herein, the term “disease-associated antigen” means either thatthe antigen is not expressed in normal, healthy cells, or that theantigen is expressed in abnormal quantity in diseased cells. Existenceof disease-associated antigen on cells greatly increases the amount ofthe chimeric pathogen that attacks such disease-associated cells.

Preferably the antibody fragment is a single chain antibody (scFv), ),but Fv and CDR fragments can also be used. The antibody fragment ispreferably incorporated into the surface integrin-like protein to form afusion protein.

In a preferred embodiment, the chimeric pathogen is a chimeric Candidaalbicans, the most common fungal pathogen of humans. Inimmunocompromised individuals, C. albicans is a dangerous, and sometimeslethal pathogen. The primary protein responsible for C. albicanstargeting is an integrin-like transmembrane protein known as INT1. INT1contains an integrin-like domain (known as the I domain), which is theputative targeting region of this protein. FIGS. 1A-W show thenucleotide sequence of the gene that encodes the integrin-like INT1protein of C. Albicans (GenBank Accession # U35070) (SEQ ID NO:1).

The preferred antibody fragment for incorporation into the INT1 proteinas a fusion protein is a single chain antibody (scFv), but Fv and CDRfragments can also be used. Thus, in this embodiment, the power andspecificity of scFv antibody fragments, which now exist against aconsiderable number of cell surface targets in cancer cell lines, iscombined with the ability to not only bind to the cancer cell mass, butto invade and destroy tumors aggressively and selectively in a mannerindependent and complementary to the body's own defenses.

The present invention exploits the mechanism involved in the filamentoustransformation of C. albicans and similar pathogenic organisms, which iscontrolled by a regulatory system similar to that used by Saccharomycescerevisiae to alter its own morphology under nitrogen starvationconditions and agar invasive conditions. The evolutionary conservationof this pathway has greatly facilitated deconvoluting the biochemistryinvolved, and recent research has resulted in a better understanding ofproteins responsible for C. albicans intercellular binding andpathogenicity. In particular, INT1 is the previously known butunidentified surface protein that is strongly crossreactive withantibodies for certain leukocyte integrins, and appears to be theprimary protein responsible for attaching to target cells (C. A. Gale etal., Science (1998) 279:1355-1358). INT1 is a transmembrane surfaceprotein isolated by cDNA screening of the C. albicans genome byoligonucleotide probes derived from the conserved region of humanintegrins (C. Gale et al., Proc. Natl. Acad. Sci. USA (1996)93:357-361). Over the last fifteen years, many reports have appeared inthe literature describing surface proteins that are related toI-subunits of the leukocyte integrins IM/l2 (Mac-1; CD11b/CD18) andIX/θ2 (p150,95; CD11c/CD18) (C. M. Bendel, J. Clin Invest. (1993)92:1840-1849 and references therein). Many monoclonal antibodies thatrecognize epitopes of these leukocyte surface proteins cross react toblastospores and germ tubes of C. albicans, sometimes with the sameaffinity as to the original human targets.

Upon ligand binding, INT1 signals cell morphology changes that inducehyphae growth. This signaling pathway appears to be largely independentof the mating factor MAPK pathway, which is most commonly associatedwith morphologic changes. Unlike the pathway that is triggered by ligandbinding, the MAPK pathway is triggered by environmental stimulation,such as changes in pH, temperature, mating signaling or nutrientavailability, and terminates in transcription factor STE12 in S.cerevisiae (Gale 1996; C. J. Gimeno et al., Cell (1992) 68, 1077-10901992 and R. L. Roberts et al., Genes Dev. (1994) 8, 2974-2985) (FIG. 4).

The second pathway to hyphal morphology, which is less wellcharacterized, depends on direct stimulation by serum. Addition ofmammalian serum to an otherwise spheroplast culture of C. albicansinduces hyphae growth, even when the signaling cascade terminating inSTE12 (briefly described above) is completely knocked out (Lo 1997).INT1 instead communicates morphology changes via a second, STE12independent, pathway that appears to have as an intermediary proteinAsh1. Ash 1 is a daughter cell-specific protein that helps regulatefilamentous growth, and may interact with STE20 (S. Chandarlapaty etal., Mol. Cell Biol. (1998) 18, 2884-2891). This pathway terminates atthe transcriptional level in a bHLH class protein known as PHD1 recentlyisolated in S. cerevisiae. The homologous protein in C. albicans isknown as EFG1. The sequence for EFG1 is fully described in W. R. Stoldtet al., EMBO Journal (1997) 16, 1982-1991, which is incorporated hereinby reference in its entirety.

For both S. cerevisiae and C albicans, overexpression of theirrespective analog is sufficient to induce hyphae growth (Stoldt 1997).Importantly, elimination of filamentous growth can only be achievedafter disabling both of these two pathways. This observation also rulesout a third pathway for signaling.

In construction of the illustrative C. albicans chimeric pathogen, theintegrin homolog INT1 was isolated using conserved transmembranesequences from mammalian integrins to clone the cDNA copy of the gene inC. albicans. Removal of the INT1 protein reduces specific adhesion of C.albicans to HeLa cells by 39%. Therefore, though INT1 is a criticalprotein for binding to a target, other proteins as well must serve tohelp mediate this interaction (vide infra), though these proteins haveyet to be identified. Still, previous research appears to be consistentwith cell surface binding by a single, integrin-like protein ofapproximate MW of 165 kDa. Interestingly, transgenic experiments whereINT1 was overexpressed on the surface of Saccharomyces cerevisiae, anonadhesive and nonpathogenic species, caused strong adhesion tomammalian cells. Thus, INT1 alone is sufficient for target binding.

Deletion of the INT1 gene cripples filamentous growth of Candida, thoughnot entirely eliminating it. It has been shown that invasive growth ofthis type is necessary for parasitic microorganisms to successfullyinvade host tissues (W-S Lo et al., Mol. Biol. Cell (1998) 9:161-171).In vivo testing of the pathogenicity of an INT1 of pathogenicity of theINT1 deletion strain of C. albicans on mice was conducted and showed adramatic reduction in mouse lethality compared to wild-type strains(Gale 1996).

The similarity of INT1 in C. albicans to mammalian integrins is notlimited to antibody cross reactivity and sequence similarity in thetransmembrane region. Notably, INT1 also appears to contain numerousmotifs similar by homology to mammalian integrin motifs. These include(1) two FE-hand divalent cation binding sites that likely mediate targetbinding; (2) a single cytosolic tyrosine for kinase signaling; and, mostimportantly, (3) a region that appears to be homologous to the I domainof integrins. Similar to the higher mammalian IM and IX that recognizeiC3b and fibrinogen, the I domain like region in C. albicans INT1 isgenerally thought to be the binding site that targets iC3b. This isfurther supported by its ˜25% sequence identity with the fibrogenbinding domain of Staphylococcus aureus.

In the illustrative preferred embodiment of the invention chimericpathogen, the I domain of the wild-type INT1 protein, whichnonspecifically targets fibrinogen, is replaced with an scFv thattargets cancer cells. Many scFvs already have been developed that bindto a wide variety of tumor cells for therapeutic applications. Suchstudies take advantage of the severe misregulation of surface proteinpopulations in tumors by utilizing scFvs that bind epitopes found insuch surface proteins. For example, therapeutic applications involvingT-cell, viral, and/or drug targeting has already been proven in vivousing scFvs shown in Table 1 below.

TABLE 1 CANCER LINE ANTIBODY ANTIGEN AND LOCATION REFERENCES OTHER CC49TAG-72 Adenocarcinoma McGuiness 1999 (colon, Shu 1993 ovarian, breast)Kashmiri 1995 FRP5 ERBB2 Breast, ovarian Moritz 1994 Previously used toHarwerth 1992 construct cytotoxic Hynes 1993 C-lymphocytes. Also used todirect virus targeting (Galmiche 1997) GA733.2 EGP-2 VariousRen-Heidenreich 2000 HMN-14 CEA Colorectal, breast, Nolan 1999Previously used to pancreas, other construct killer T-cells VFF17 CD44Cervical cancer, Dall 1997 lymph metastases Hekele 1996 MOV19 I-FRNonmucinous ovarian Melani 1998 carcinoma 7.16.4 Neu Breast Katsumata1995 Antigen (neu) is same Stankovski 1993 as ERBB2, and is Disis 1997protein bound by Herceptin. MLuCl L(Y) TAA Various Mezzanzanica Targetsmisregulated 1998 carbohydrates. Lewis (Y) tumor associated antigen

By replacing the I domain in the integrin-like surface protein with ascFv that binds to a disease-associated tumor cell, the inventionchimeric pathogenic organisms are engineered to take advantage of theseverely misregulated production of surface protein populations intumors. In the present invention, the antibody fragment, preferably as ascFv, is incorporated into the position of the native binding domain ofan integrin-like protein (i.e., the creation of a fusion protein thatcontains the scFv incorporated in the place of the I domain in thewild-type pathogenic organism). Many antibody fragments have alreadybeen tested for selective binding to a known tumor-associated antigen,for example, as shown in Table 1. Representative non-limiting examplesof tumor associated antigens to which scFvs of the invention chimericpathogens bind include GAG-72, ERBB2, EGP-2, CEA, CD44, I-FR, neu, theLewis (Y) tumor associated antigen, and the like.

As used herein, the terms “disease- or tumor-associated antigen” and“disease- or tumor-associated epitope” encompass antigens and epitopes,respectively, found in surface proteins produced in large amounts invarious types of tumors as well as various types of marker proteins (andthe epitopes contained therein) that are found associated with tumorcells and not found associated with normal cells. Representativenon-limiting examples of tumors having associated antigens to whichantibody fragments (e.g., scFvs) of the invention chimeric pathogensbind includes adenocarcinoma of colon, ovary or breast; cervical cancer,nonmucinous ovarian carcinoma; breast, ovarian, colorectal, andpancreatic cancer, and the like. Invention chimeric pathogenic organismsare incapable of infiltrating a cell in the subject until the antibodyfragment in the chimeric integrin-like protein binds to its targetepitope, triggering a virulent transformation of the chimeric pathogenicorganism. Therefore, the invention chimeric pathogenic organisms aresubstantially incapable of pathogenic activity, such as infiltration, ofcells other than their target cells (e.g., cancer cells).

Preferably, the antibody fragment is a scFv and is introduced in theplace of the I domain of INT-1 in C. albicans. Once engineered toreplace the wild-type binding domain of the INT1 protein with an antigenbinding region (e.g. scFv) from cancer-specific antibodies, theinvention mutant C. albicans strain will specifically bind to a cancerline dictated by the targeting of the scFv-INT1 fusion protein.

Optionally, in order to direct pathogenicity specifically to the targetcell (e.g., a carcinoma cell) a gene in the pathogenic organism fromwhich the chimeric organism is derived that is required for invasivegrowth is disabled or removed and a DNA construct comprising areengineered copy of the gene necessary for invasive growth isintroduced into the chimeric organism under the regulatory control of atranscription factor that regulates filamentous transformation of theorganism. However, the gene removed should be one that does notsignificantly affect vegetative growth of the organism so that largequantities of this chimeric organism can be produced using standardculture techniques.

For example, in C albicans, the wild-type gene is placed under thecontrol of a EFG1p response element. While the CaFTR1 gene is currentlypreferred for reengineering in C. albicans, those of skill in the artcan readily substitute for reengineering (i.e., in the place of theCaFTR1 gene) another gene from the pathogenic organism that is essentialor preferred for pathogenic invasion.

Preferably, in the invention chimeric C. albicans, the wild-type CAFTRgene is either disabled or removed and a DNA construct comprising awild-type CAFTR gene under the control of a EFG1p response element isintroduced. Overexpression of EFG1 in C. albicans leads to enhancedfilamentous growth in liquid and on solid media. Overexpression of EFG1by a PCK1p-EFG1 fusion is described by A. Sonneborn, Infect Immun (1999)67:9:4655-60, which is incorporated herein by reference in its entirety(See also, V. R. Stoldt et al., EMBO J (1997) 16:8 1982-91). Thenucleotide sequence for the CaFTR1 gene of C. albicans is found at NCBIGenBank Number AF195775.

CaFTR1 extracts iron from mammalian tissues that withhold metals frommicrobial predators as a defense mechanism (D. M. DeSilva et al.,Physiol. Rev. (1996) 76, 31-47 and H. Gunshin et al., Nature (1997) 388,482-488). Removal of the native CaFTR1 completely abrogatespathogenicity. Mice injected with a mutant C. albicans having a disabledCaFTR1 gene survive entirely; while those injected with an equal amountof wild-type C. albicans do not. Under circumstances of normalunicellular growth in an abundance of iron, though, CaFTR1 is not anessential gene. In conditions where iron is in limited quantities, forinstance during circulation through a host designed to have limitingnutrient levels, this gene is highly upregulated. Removal of the CaFTR1gene only causes a growth (and thus invasion) deficiency whenpathogenesis is initiated. This protein is normally regulated entirelyindependently from the morphology signaling pathway, and itsconcentration is dependent only on the heavy metals detected in theenvironment. By placing this protein under the transcriptional controlof the cell morphology pathway initiated by INT1, as described herein,the pathogenicity of the overall assembly can be tightly restricted toscFv-INT1 targeted cells.

In this preferred embodiment of the invention chimeric pathogenicorganism, binding of the antibody to the disease-associated antigentriggers both expression of the CAFTR gene in the DNA construct andfilamentous transformation of the chimeric pathogenic C. albicans. Sinceexpression of INT1 in wild-type C. albicans is activated by INT1 bindingto other cells, placing expression of the C. albicans iron transporterunder control of the Efg1 expression system ensures that both thepathogenicity and the binding of the invention chimeric organism isdirected specifically to target cells. If the antibody fragmentincorporated in the place of the INT1 binding domains is specific for atumor cell, the pathogenicity of the C. albicans cell line is directedspecifically towards target cancerous cells, and nonspecific toxicity isinhibited. In other words, the virulence of the engineered strain of C.albicans will only be activated once scFv-INT1 binds to its targetantigen on the surface of the carcinoma.

The gene that triggers filamentous growth can be disabled in theinvention chimeric organism using any method known in the art, forexample, by disruption of the gene at both diploid loci using standardtechniques. The native gene can be reintroduced under the control of aresponse element (e.g., a transcription factor) that regulatesfilamentous transformation of the organism using known techniques, suchas by use of homologous recombination (as described in Example 1herein). This regulatory reassignment of the gene that triggerstransformation tightly limits the pathogenicity dependent on thisprotein to the specified target.

In yet another embodiment, the present invention provides methods fortreating a disease associated with the presence of cells having adisease-associated surface antigen in a subject in need thereof. Theinvention method includes administering to the subject, atherapeutically effective amount of an invention chimeric pathogenicorganism so as to cause binding of the antibody fragment to thedisease-associated antigen on the cell, thereby specifically treatingthe disease by triggering infiltration of the chimeric pathogenicorganism into the cells without substantial damage to healthy cells. Theinvention method may further include administering to the subject animmunosuppressive agent to inhibit the subject's immune system fromdestroying the chimeric pathogen prior to achieving a therapeuticeffect. Representative immunosuppressive agents useful in the practiceof the invention methods include such agents as cyclosporin A, OKT3,FK506, mycophenolate mofetil (MMF), azathioprine, corticosteroids (suchas prednisone), antilymphocyte globulin, antithymocyte globulin, and thelike. In a preferred embodiment of the invention methods, the inventionchimeric pathogen is used to target and attack tumor cells.

In invention therapeutic methods, the chimeric pathogenic organisms areused to infiltrate and destroy both ex vivo cell lines (e.g., tumor celllines), as well as in vivo murine models of human carcinomas, and thelike. The invention pathogenic organisms also have specific utility asresearch reagents for the testing of therapeutic compositions. Forexample, the invention chimeric organisms can be used to compare thetherapeutic effect against a particular cell line of various antibodyfragments engineered into the surface integrin-like protein. Binding ofinvention organisms to these ex vivo and in vivo models can be testedfor efficacy using known assays (e.g., mouse tumor models) to determinebinding of the antibody fragment (e.g., single chain antibody) to thetarget antigen on a disease-associated cell.

The chimeric pathogens used in practice of the invention method can beadministered for therapeutic purposes, such as treatment of tumor, byany route known to those of skill in the art, such as intraarticularly,intracisternally, intraocularly, intraventricularly, intrathecally,intravenously, intramuscularly, intraperitoneally, intradermally,intratracheally, intracavitarily, and the like, as well as by anycombination of any two or more thereof.

The most suitable route for administration will vary depending upon thedisease state to be treated, or the location of the suspected conditionor tumor to be treated. For example, for treatment of inflammatoryconditions and various tumors, local administration, includingadministration by injection directly into the body part containing thetumor provides the advantage that the chimeric pathogen can beadministered in a high concentration without risk of the complicationsthat may accompany systemic administration thereof.

The chimeric pathogen is administered in “a therapeutically effectiveamount.” An effective amount is the quantity of a chimeric pathogennecessary to aid in treatment, inhibition or destruction of diseasedtissue (e.g. tumor) under treatment in a subject. A “subject” as theterm is used herein is contemplated to include any mammal, such as adomesticated pet, farm animal, or zoo animal, but preferably is a human.Amounts effective for therapeutic use will, of course, depend on suchfactors as the size and location of the body part to be treated, theaffinity of the antibody fragment for the target antigen, the type oftarget tissue, as well as the route of administration. Localadministration of the targeting construct will typically require asmaller dosage than any mode of systemic administration, although thelocal concentration of the chimeric pathogen may, in some cases, behigher following local administration than can be achieved with safetyupon systemic administration.

The invention composition can also be formulated as a sterile injectablesuspension according to known methods using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1-4, butanediol. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil maybe employed, including synthetic mono- or diglycerides, fatty acids(including oleic acid), naturally occurring vegetable oils like sesameoil, coconut oil, peanut oil, cottonseed oil, etc., or synthetic fattyvehicles like ethyl oleate, or the like. Buffers, preservatives,antioxidants, and the like, can be incorporated as required, or,alternatively, can comprise the formulation.

Preferably the antibody fragment is a scFv incorporated into thechimeric surface protein of the pathogen as a targeting device and isnot relied upon as the toxic agent. Rather, it is the pathogenicorganism itself that invades and destroys the target cells in accordancewith the present invention. A single chain antibody (scFv) is agenetically engineered molecule containing the variable region of thelight chain and the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule. Methods of making these fragments are known in the art. (Seefor example, Harlow & Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, 1988, incorporated herein by reference). Asused in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three-dimensional structural characteristics, aswell as specific charge characteristics.

Fv fragments comprise an association of V_(H) and V_(L) chains. Thisassociation may be noncovalent, as described in Inbar et al., Proc.Nat'l Acad. Sci. USA 69:2659, 1972. Alternatively, the variable chainscan be linked by an intermolecular disulfide bond or cross-linked bychemicals such as glutaraldehyde. See, e.g., Sandhu, Crit. Rev. Biotech.12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993. Preferably,the Fv fragments comprise V_(H) and V_(L) chains connected by a peptidelinker. These single-chain antigen binding proteins (scFv) are preparedby constructing a structural gene comprising DNA sequences encoding theV_(H) and V_(L) domains connected by an oligonucleotide. The structuralgene is inserted into an expression vector, which is subsequentlyintroduced into a host cell such as E. coli. The recombinant host cellssynthesize a single polypeptide chain with a linker peptide bridging thetwo V domains. Methods for producing scFvs are described, for example,by Whitlow et al., Methods: a Companion to Methods in Enzymology, 2: 97,1991; Bird et al., Science 242:423-426, 1988; Pack et al.,Bio/Technology 11:1271-77, 1993; Sandhu, supra, and Ladner et al., U.S.Pat. No. 4,946,778, which is hereby incorporated by reference in itsentirety.

Another form of an antibody fragment suitable for incorporation as afusion protein in invention chimeric pathogenic organisms is a peptidecoding for a single complementarity-determining region (CDR). CDRpeptides (“minimal recognition units”) can be obtained by constructinggenes encoding the CDR of an antibody of interest. Such genes areprepared, for example, by using the polymerase chain reaction tosynthesize the variable region from RNA of antibody-producing cells.See, for example, Larrick et al., Methods: a Companion to Methods inEnzymology, 2: 106, 1991.

Antibodies that bind to a tumor cell or other disease-associated antigencan be prepared using an intact polypeptide or biologically functionalfragment containing small peptides of interest as the immunizingantigen. The polypeptide or a peptide used to immunize an animal(derived, for example, from translated cDNA or chemical synthesis) canbe conjugated to a carrier protein, if desired. Commonly used carriersthat are chemically coupled to the peptide include keyhole limpethemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanustoxoid, and the like. The coupled peptide is then used to immunize theanimal (e.g., a mouse, a rat, or a rabbit).

The preparation of such monoclonal antibodies is conventional. See, forexample, Kohler & Milstein, Nature 256:495, 1975; Coligan et al.,sections 2.5.1-2.6.7; and Harlow et al., in: Antibodies: a LaboratoryManual, page 726 (Cold Spring Harbor Pub., 1988), which are herebyincorporated by reference. Briefly, monoclonal antibodies can beobtained by injecting mice with a composition comprising an antigen,verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B lymphocytes, fusing the Blymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones that produce antibodies to theantigen, and isolating the antibodies from the hybridoma cultures.Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3;Barnes et al., Purification of Immunoglobulin G (IgG), in: Methods inMolecular Biology, Vol. 10, pages 79-104 (Humana Press, 1992).

Antibodies of the present invention may also be derived from subhumanprimate antibodies. General techniques for raising therapeuticallyuseful antibodies in baboons can be found, for example, in Goldenberg etal., International Patent Publication WO 91/11465 (1991) and Losman etal., 1990, Int. J. Cancer 46:310, which are hereby incorporated byreference. Alternatively, a therapeutically useful antibody may bederived from a “humanized” monoclonal antibody. Humanized monoclonalantibodies are produced by transferring mouse complementaritydetermining regions from heavy and light variable chains of the mouseimmunoglobulin into a human variable domain, and then substituting humanresidues in the framework regions of the murine counterparts. The use ofantibody components derived from humanized monoclonal antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions. General techniques for cloning murine immunoglobulinvariable domains are described, for example, by Orlandi et al., Proc.Nat'l Acad. Sci. USA 86:3833,1989, which is hereby incorporated in itsentirety by reference. Techniques for producing humanized monoclonalantibodies are described, for example, by Jones et al., Nature 321:522,1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science239:1534, 1988; Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285, 1992;Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer et al., J. Immunol.150:2844, 1993, which are hereby incorporated by reference.

It is also possible to use anti-idiotype technology to producemonoclonal antibodies, which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hyper variable region that is the“image” of the epitope bound by the first monoclonal antibody.

The assembly, selection, and integration of these chimeric scFv-INT1products are conducted using standard molecular biology, for example asis described in Example 1 herein. The proper assembly of the inventionchimeric scFv-INT-1 protein and adhesion to the epitopes in target celllines, e.g., tumor cell lines, can be tested by introduction of thechimeric assembly into S. cerevisiae, preferably under the control of apromoter, such as the actin promoter, that is constantly activated insuch yeast cell lines. Yeast cells (e.g., Saccharomyces cerevisiae)possess an efficient and precise system for genetic recombination. Thenatural process of homologous recombination depends on a system ofenzymes that search for regions of sequence homology between two DNAmolecules (which may be entire chromosomes). Once homology is found, anexchange of information is possible.

Plasmids or other vectors carrying recombinant-DNA (r-DNA) clones whichcontain naturally-occurring yeast sequences and which are introducedinto cells by standard transformation methods are capable of stablyintegrating into the yeast genome at sites of homology. The efficiencyof this process can be increased by up to a thousand-fold by introducinga double-strand break within a DNA sequence on the incoming DNA moleculethat is homologous to a sequence resident in the yeast cell. The clonedyeast DNA on the transforming vector is referred to herein as thetargeting sequence, and the site of integration is referred to herein asthe target site.

In one process described in U.S. Pat. No. 5,783,385 to Treco , et al.,which is incorporated herein by reference in its entirety, a targetingDNA molecule, e.g., a bacterial plasmid, which is non-replicating inyeast is introduced into the population of host yeast cells containingthe r-DNA. The bacterial plasmid has a selectable marker gene thatfunctions in yeast and a first targeting DNA sequence which ishomologous in part to a second target r-DNA clone sequence.Preferentially, the targeting plasmid is cut with a restrictionendonuclease that introduces a double-strand break within the targetingsequence, thereby linearizing the bacterial plasmid and providing DNAends which are recombinogenic to stimulate the process of homologousrecombination with host yeast sequences. Because the plasmid isnon-replicating in yeast, stable transformation with the selectablemarker can only proceed by homologous recombination. The efficiency oftransformation by homologous recombination is increased when the plasmidis cut by restriction enzyme digestion within the targeting DNA sequencehomologous in part to the target r-DNA sequence.

The host yeast cells are grown under conditions such that only thoseyeast cells that have been stably transformed, i.e., have had theplasmid and selectable marker stably integrated in the host cell byhomologous recombination will be able to grow. In a correctly targetedevent, the entire plasmid is stably incorporated contained in the hostyeast cell by homologous recombination of the targeting DNA sequence ofthe plasmid and the homologous target r-DNA clone sequence. Only thosefew host yeast cells that contain the desired, target r-DNA clonesequence (and have thereby undergone homologous recombination with thetargeting plasmid) are able to grow under the new growth conditions, dueto the introduction of the yeast-selectable marker gene contained on thetargeting plasmid.

The vast majority of the population of the host yeast cells containingr-DNA clone sequences that are not homologous to the targeting DNAsequence contained on the plasmid, do not have the plasmid incorporatedby homologous recombination and, therefore, do not acquire the markergene that is essential for growth under the selection conditions.Therefore, it is preferable that any yeast-selectable marker gene thatis contained on the incoming targeting plasmid has been deleted entirelyor almost entirely from the genome of the host yeast strain that is usedfor the vector. This prevents any spurious homologous recombinationevents between the incoming yeast-selectable marker gene and any othernatural yeast genetic loci. If a yeast-selectable marker gene on theincoming targeting plasmid is not deleted from the yeast genome, but isretained as a mutated, non-functional portion of the yeast chromosome,more positive scores for homologous recombination will have to bescreened to ensure that the homologous recombination event has takenplace between the targeting DNA sequence on the bacterial plasmid andthe desired, target r-DNA clone sequence. Cells with the integratedmarker can grow into colonies when plated on appropriate selectivemedia.

Alternatively, a yeast-selectable marker gene on the incoming targetingDNA molecule can be a bacterial gene that confers drug resistance toyeast cells, e.g., the CAT or neo genes from Tn9 and Tn903, or bacterialamino acid or amino acid nucleoside prototrophy genes, e.g., the E. coliargH, trpC, and pyrF genes.

Methods for plasmid purification, restriction enzyme digestion ofplasmid DNA and gel electrophoresis, use of DNA modifying enzymes,ligation, transformation of bacteria, transformation of yeast by thelithium acetate method, preparation and Southern blot analysis of yeastDNA, tetrad analysis of yeast, preparation of liquid and solid media forthe growth of E. coli and yeast, and all standard molecular biologicaland microbiological techniques can be carried out essentially asdescribed in Ausubel et al. (Ausubel, F. M. et al., Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley-Interscience,New York, 1987).

Once the proper assembly of the invention chimeric scFv-INT-1 proteinand adhesion to the epitopes in target cell lines, e.g., tumor celllines, has been tested in a non-pathogenic yeast cell (e.g.,Saccharomyces cerevisiae) homologous recombination can be used to inserta polynucleotide sequence encoding the chimeric scFv-INT1 into Candidaalbicans, and similar ex vivo experiments as those performed for S.cerevisiae will be performed to assure that replacement of the I domaindoes not seriously impair the proper folding and targeting of scFv-INT1.At this point, ex vivo experiments verifying adhesion of this mutant C.albicans strain to cancer cells are performed, in addition topreliminary in vivo mice experiments to ascertain that this targetingalone is adequate in mice to restrict pathogenicity and targeting totumors.

The most common model for human cancers is a murine subject that hasbeen transfected with human carcinomas. After an incubation periodvarying from weeks to months after carcinoma introduction to allowgrowth of test tumors, transfected mice will be treated with thegenetically modified C. albicans. Survival of the mice and tumorspreading are monitored over time. Biopsies of the tumorous tissues canalso be taken to investigate C. albicans invasion. By using large groupsof genetically identical mice, aggregate data can be collected.

Evolution has optimized certain organisms to invade mammalian tissue.The present invention harnesses this powerful and highly pathogenictrait to generate a new weapon against cancer and other diseasescharacterized by the presence of cells with a disease-associatedantigen. In contrast to more indirect methodologies previously appliedthat recruit the natural immune system responses, fusion scFv-INT1proteins targeted to disease-associated tissues will direct aggressiveinvasion of the naturally invasive pathogen to diseased host tissue. Themethod of the present invention is a novel approach to cancer treatmentthat recruits the previously untapped resource of pathogenic organisms(e.g. fungi) as potent and specific therapy to eliminate diseased tissuecharacterized by disease-associated antigens.

The invention will now be described by reference to the followingnon-limiting illustrative example:

EXAMPLE 1 Construction of the scFv-INT1 Fusion Gene

Using bulk genomic DNA from C. albicans, primers 1 and 2 (shown in FIGS.2A-B) (SEQ ID NOS:2 and 3) are used for PCR amplification of the INT1gene (available from GenBank under accession number U35070) (SEQ IDNO:1) as previously described (Gale 1996). These primers insert SacI andApaI restriction sites at the 5′ and 3′ ends of the coding region ofINT1, respectively. These restriction sites are both nonexistent in theORF of the gene (see gene sequence in FIGS. 1A-W). The 5 kB product ofthis PCR reaction is isolated using a standard Qiagen desalting kit,digested with the appropriate enzymes SacI and ApaI, and ligated intopredigested and dephosphorylated pBluescript II SK (+) phagemid plasmidaccording to the manufacturer's instructions (Product #212205,Stratagene, LaJolla, Calif.). ssDNA incorporating the INT1 gene is thengenerated using standard techniques with helper phage and uridine indut⁻ ung⁻ strains of E. Coli according to the manufacturer'sinstructions.

To introduce multiple cloning sites in the ssDNA PCR product in theplace of the I domain of INT1, primer 7 (shown in FIGS. 2A-B) (SEQ IDNO:8) is used in a standard polymerase/ligase reaction; also thuseliminating the I domain. Isolation of the generated plasmids isperformed using standard techniques.

Single chain antibodies (scFvs) having the target antigen binding regionof a desired antigen are generated using reverse transcriptase PCR ofthe bulk RNA from antibody-generating cell lines using primers 4 to 6(shown in FIGS. 2A-B) (SEQ ID NOS:5, 6, and 7). The binding regions aresubcloned into the cut and dephosphorylated plasmid prepared asdescribed above, and then a fusion gene is isolated and characterizedusing techniques described in Z. Eshhar et al., Methods in Enzymology8:133-142 (1995), except that Primers 4 to 6 differ from those shown inEshhar by including different restriction endonuclease sites, as INT1has restriction sites for the nucleases used by Eshhar. The primers usedto remove the binding regions of the heavy and light chains incorporatea linker that allows the now-assembled scFv-INT1 protein to have thebinding region activated and folded properly.

The chimeric INT1-scFv fusion protein is directly expressed in E. Colifor in vitro studies of folding and binding using known techniquesdescribed in Sections 10.0.1 and 16.1 to 16.7 of Current Protocols inMolecular Biology, Collected Volumes 1 to 4, edited by Ausubel, F. M. etal., John Wiley &Sons, 2000. In addition, the chimeric INT1-scFv fusionprotein is incorporated into S. Cerevisiae using an expression plasmidcontaining the nucleotide sequence that encodes the fusion protein forcell-cell studies and is incorporated back into Candida albicans byhomologous recombination using techniques described in Section 13.10.3of Current Protocols in Molecular Biology, supra. Thus, a tumor-specificorganism is readily accomplished.

It will be apparent to those skilled in the art that various changes maybe made in the invention without departing from the spirit and scopethereof, and therefore, the invention encompasses embodiments inaddition to those specifically disclosed in the specification, but onlyas indicated in the appended claims.

1. A method for generating a chimeric Candida organism from a pathogenicorganism that possesses in the wild-type an INT1 protein with an Idomain, said method comprising: replacing the I domain in the INT1protein of the pathogenic organism with an antibody fragment that bindsto a disease-associated antigen on a diseased cell; wherein thewild-type pathogenic organism undergoes virulent transformation bybinding of the I domain of the surface INT1 protein to a cell, andwherein the chimeric Candida organism undergoes virulent transformationby binding of the antibody fragment to the disease-associated antigen onthe cell.
 2. The method of claim 1, wherein the pathogenic organism isC. albicans and wherein the method further comprises disabling thewild-type high-affinity iron transporter gene in the C. albicans, andintroducing a DNA construct comprising a wild-type high-affinity irontransporter gene under the control of a EFG1p response element, whereinbinding of the antibody fragment to the disease-associated antigentriggers expression of the high-affinity iron transporter gene in theDNA construct and filamentous transformation in the chimeric pathogenicC. albicans.
 3. The method of claim 2, wherein the antibody fragment isa single chain antibody.
 4. The method of claim 2, wherein the antibodyfragment binds to an antigen on a tumor cell.
 5. The method of claim 4,wherein the disease-associated antigen is contained in an abnormalsurface protein of the tumor cell.